Methods and compositions for the correlation of single nucleotide polymorphisms in the vitamin K epoxide reductase gene and warfarin dosage

ABSTRACT

The present invention provides a method of identifying a human subject having increased or decreased sensitivity to warfarin, comprising detecting in the subject the presence of a single nucleotide polymorphism in the VKOR gene, wherein the single nucleotide polymorphism is correlated with increased or decreased sensitivity to warfarin, thereby identifying the subject having increased or decreased sensitivity to warfarin.

STATEMENT OF PRIORITY

The present application is a 35 U.S.C. § 371 national phase applicationof PCT International Application No. PCT/US2004/031481, having aninternational filing date of Sep. 23, 2004, which claims the benefit,under 35 U.S.C. § 119(e), of U.S. Provisional Application Ser. No.60/505,527, filed Sep. 23, 2003, the entire contents of each of whichare incorporated by reference herein.

GOVERNMENT SUPPORT

The present invention was made, in part, with the support of grantnumbers 5P01 HL06350-42 and 5-R01 HL48318 from the National Institutesof Health. The United States Government has certain rights to thisinvention.

FIELD OF THE INVENTION

The present invention concerns isolated nucleic acids, host cellscontaining the same, and methods of use thereof, as well as methods andcompositions directed to identification of single nucleotidepolymorphisms (SNPs) in the Vitamin K epoxide reductase (VKOR) gene andtheir correlation with sensitivity to warfarin.

BACKGROUND OF THE INVENTION

The function of numerous proteins requires the modification of multipleglutamic acid residues to γ-carboxyglutamate. Among these vitaminK-dependent (VKD) coagulation proteins, FIX (Christmas factor), FVII,and prothrombin are the best known. The observation that a knock-out ofthe gene for matrix Gla protein results in calcification of the mouse'sarteries (Luo et al. (1997) “Spontaneous calcification of arteries andcartilage in mice lacking matrix GLA protein” Nature 386:78-81)emphasizes the importance of the vitamin K cycle for proteins withfunctions other than coagulation. Moreover, Gas6 and other Gla proteinsof unknown function are expressed in neural tissue and warfarin exposurein utero results in mental retardation and facial abnormalities. This isconsistent with the observation that the expression of VKD carboxylase,the enzyme that accomplishes the Gla modification, is temporallyregulated in a tissue-specific manner with high expression in thenervous system during early embryonic stages. Concomitant withcarboxylation, reduced vitamin K, a co-substrate of the reaction, isconverted to vitamin K epoxide. Because the amount of vitamin K in thehuman diet is limited, vitamin K epoxide must be converted back tovitamin K by vitamin K epoxide reductase (VKOR) to prevent itsdepletion. Warfarin, the most widely used anticoagulation drug, targetsVKOR and prevents the regeneration of vitamin K. The consequence is adecrease in the concentration of reduced vitamin K, which results in areduced rate of carboxylation by the γ-glutamyl carboxylase and in theproduction of undercarboxylated vitamin K-dependent proteins.

In the United States alone, warfarin is prescribed to more than onemillion patients per year and in Holland, it has been reported thatapproximately 2% of the population is on long term warfarin therapy.Because the dose of warfarin required for a therapeutic level ofanticoagulation varies greatly between patients, the utilization ofwarfarin is accompanied by a significant risk of side effects. Forexample, it has been reported that following initiation of warfarintherapy, major bleeding episodes occurred in 1-2% of patients and deathoccurred in 0.1-0.7% of patients. In spite of the dangers, it has beenestimated that warfarin use can prevent 20 strokes per induced bleedingepisode and is probably underutilized because of the fear of inducedbleeding.

The present invention overcomes previous shortcomings in the art byproviding methods and compositions for correlating single nucleotidepolymorphisms in a subject with an increased or decreased sensitivity towarfarin, thereby allowing for more accurate and rapid determination oftherapeutic and maintenance doses of warfarin at reduced risk to thesubject.

SUMMARY OF THE INVENTION

The present invention provides a method of identifying a human subjecthaving increased or decreased sensitivity to warfarin, comprisingdetecting in the subject the presence of a single nucleotidepolymorphism in the VKOR gene, wherein the single nucleotidepolymorphism is correlated with increased or decreased sensitivity towarfarin, thereby identifying the subject having increased or decreasedsensitivity to warfarin.

Additionally provided is a method of identifying a human subject havingincreased or decreased sensitivity to warfarin, comprising: a)correlating the presence of a single nucleotide polymorphism in the VKORgene with increased or decreased sensitivity to warfarin; and b)detecting the single nucleotide polymorphism of step (a) in the subject,thereby identifying a subject having increased or decreased sensitivityto warfarin.

In a further embodiment, the present invention provides a method ofidentifying a single nucleotide polymorphism in the VKOR gene correlatedwith increased or decreased sensitivity to warfarin, comprising:

a) identifying a subject having increased or decreased sensitivity towarfarin;

b) detecting in the subject the presence of a single nucleotidepolymorphism in the VKOR gene; and

c) correlating the presence of the single nucleotide polymorphism ofstep (b) with the increased or decreased sensitivity to warfarin in thesubject, thereby identifying a single nucleotide polymorphism in theVKOR gene correlated with increased or decreased sensitivity towarfarin.

In addition, the present invention provides a method of correlating asingle nucleotide polymorphism in the VKOR gene of a subject withincreased or decreased sensitivity to warfarin, comprising: a)identifying a subject having increased or decreased sensitivity towarfarin; b) determining the nucleotide sequence of the VKOR gene of thesubject of (a); c) comparing the nucleotide sequence of step (b) withthe wild type nucleotide sequence of the VKOR gene; d) detecting asingle nucleotide polymorphism in the nucleotide sequence of (b); and e)correlating the single nucleotide polymorphism of (d) with increased ordecreased sensitivity to warfarin in the subject of (a).

A further aspect of the present invention is an isolated nucleic acidencoding vitamin K epoxide reductase (VKOR), particularly mammalian(e.g., human, ovine, bovine, monkey, etc.) VKOR. Examples include (a)nucleic acids as disclosed herein, such as isolated nucleic acids havingthe nucleotide sequence as set forth in SEQ ID NO: 8 or SEQ ID NO: 9;(b) nucleic acids that hybridize to isolated nucleic acids of (a) aboveor the complement thereof (e.g., under stringent conditions), and/orhave substantial sequence identity to nucleic acids of (a) above (e.g.,are 80, 85, 90 95 or 99% identical to nucleic acids of (a) above), andencode a VKOR; and (c) nucleic acids that differ from the nucleic acidsof (a) or (b) above due to the degeneracy of the genetic code, but codefor a VKOR encoded by a nucleic acid of (a) or (b) above.

The term “stringent” as used here refers to hybridization conditionsthat are commonly understood in the art to define the commodities of thehybridization procedure. Stringency conditions can be low, high ormedium, as those terms are commonly know in the art and well recognizedby one of ordinary skill. High stringency hybridization conditions thatwill permit homologous nucleotide sequences to hybridize to a nucleotidesequence as given herein are well known in the art. As one example,hybridization of such sequences to the nucleic acid molecules disclosedherein can be carried out in 25% formamide, 5×SSC, 5× Denhardt'ssolution and 5% dextran sulfate at 42° C., with wash conditions of 25%formamide, 5×SSC and 0.1% SDS at 42° C., to allow hybridization ofsequences of about 60% homology. Another example includes hybridizationconditions of 6×SSC, 0.1% SDS at about 45° C., followed by washconditions of 0.2×SSC, 0.1% SDS at 50-65° C. Another example ofstringent conditions is represented by a wash stringency of 0.3 M NaCl,0.03M sodium citrate, 0.1% SDS at 60-70° C. using a standardhybridization assay (see SAMBROOK et al., EDS., MOLECULAR CLONING: ALABORATORY MANUAL 2d ed. (Cold Spring Harbor, N.Y. 1989, the entirecontents of which are incorporated by reference herein). In variousembodiments, stringent conditions can include, for example, highlystringent (i.e., high stringency) conditions (e.g., hybridization tofilter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mMEDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C.), and/ormoderately stringent (i.e., medium stringency) conditions (e.g., washingin 0.2×SSC/0.1% SDS at42° C.).

An additional aspect of the present invention is a recombinant nucleicacid comprising a nucleic acid encoding vitamin K epoxide reductase asdescribed herein operatively associated with a heterologous promoter.

A further aspect of the present invention is a cell that contains andexpresses a recombinant nucleic acid as described above. Suitable cellsinclude plant, animal, mammal, insect, yeast and bacterial cells.

A further aspect of the present invention is an oligonucleotide thathybridizes to an isolated nucleic acid encoding VKOR as describedherein.

A further aspect of the present invention is isolated and purified VKOR(e.g., VKOR purified to homogeneity) encoded by a nucleic acid asdescribed herein. For example, the VKOR of this invention can comprisethe amino acid sequence as set forth in SEQ ID NO:10.

A further aspect of the present invention is a method of making avitamin K dependent protein which comprises culturing a host cell thatexpresses a nucleic acid encoding a vitamin K dependent protein in thepresence of vitamin K and produces a vitamin K dependent protein, andthen harvesting the vitamin K dependent protein from the culture, thehost cell containing and expressing a heterologous nucleic acid encodingvitamin K dependent carboxylase, and the host cell further containingand expressing a heterologous nucleic acid encoding vitamin K epoxidereductase (VKOR) and producing VKOR as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D Comparisons of warfarin dosages in wild type, heterozygousand homozygous subjects for SNPs vk 2581, vk3294 and vk4769, as well asa comparison of warfarin dosages in wild type and heterozygous subjectsfor P450 2Y9.

FIG. 2. For each of the 13 siRNA pools, three T7 flasks containing A549cells were transfected and VKOR activity determined after 72 h. The VKORassay used 25 μM vitamin K epoxide. One siRNA pool specific for genegi:13124769 reduced VKOR activity by 64%-70% in eight repetitions.

FIG. 3. Time course of inhibition of VKOR activity by the siRNA poolspecific for gi:13124769 in A549 cells. VKOR activity decreasedcontinuously during this time period while the level of its mRNAdecreased rapidly to about 20% of normal. 25 μM vitamin K epoxide wasused for this assay. The siRNA did not affect the activity of VKDcarboxylase or the level of lamin A/C mRNA.

FIG. 4. VKOR activity was detected when mGC_(—)11276 was expressed inSf9 insect cells. ˜1×10⁶ cells were used in this assay. Reactions wereperformed using 32 μM KO at 30° C. for 30 minutes in Buffer D. Blank Sf9cells served as a negative control and A549 cells as a reference.

FIG. 5. Inhibition of VKOR by warfarin. Reactions were performed using1.6 mg microsomal proteins made from VKOR_Sf9 cells, 60 μM KO, andvarious concentration of warfarin at 30° C. for 15 minutes in Buffer D.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “a,” “an” or “the” can mean one or more than one. Forexample, “a” cell can mean a single cell or a multiplicity of cells.

The present invention is explained in greater detail below. Thisdescription is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure which do not depart from the instant invention.Hence, the following specification is intended to illustrate someparticular embodiments of the invention, and not to exhaustively specifyall permutations, combinations and variations thereof.

The “Sequence Listing” attached hereto forms a part of the instantspecification as if fully set forth herein.

The present invention may be carried out based on the instant disclosureand further utilizing methods, components and features known in the art,including but not limited to those described in U.S. Pat. No. 5,268,275to Stafford and Wu and U.S. Pat. No. 6,531,298 to Stafford and Chang,the disclosures of which are incorporated by reference herein in theirentirety as if fully set forth herein.

As used herein, “nucleic acids” encompass both RNA and DNA, includingcDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA andchimeras of RNA and DNA. The nucleic acid may be double-stranded orsingle-stranded. Where single-stranded, the nucleic acid may be a sensestrand or an antisense strand. The nucleic acid may be synthesized usingoligonucleotide analogs or derivatives (e.g., inosine orphosphorothioate nucleotides). Such oligonucleotides can be used, forexample, to prepare nucleic acids that have altered base-pairingabilities or increased resistance to nucleases.

An “isolated nucleic acid” is a DNA or RNA that is not immediatelycontiguous with both of the coding sequences with which it isimmediately contiguous (one on the 5′ end and one on the 3′ end) in thenaturally occurring genome of the organism from which it is derived.Thus, in one embodiment, an isolated nucleic acid includes some or allof the 5′ non-coding (e.g., promoter) sequences that are immediatelycontiguous to the coding sequence. The term therefore includes, forexample, a recombinant DNA that is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g., acDNA or a genomic DNA fragment produced by PCR or restrictionendonuclease treatment), independent of other sequences. It alsoincludes a recombinant DNA that is part of a hybrid gene encoding anadditional polypeptide sequence.

The term “isolated” can refer to a nucleic acid or polypeptide that issubstantially free of cellular material, viral material, or culturemedium (when produced by recombinant DNA techniques), or chemicalprecursors or other chemicals (when chemically synthesized). Moreover,an “isolated nucleic acid fragment” is a nucleic acid fragment that isnot naturally occurring as a fragment and would not be found in thenatural state.

The term “oligonucleotide” refers to a nucleic acid sequence of at leastabout six nucleotides to about 100 nucleotides, for example, about 15 to30 nucleotides, or about 20 to 25 nucleotides, which can be used, forexample, as a primer in a PCR amplification or as a probe in ahybridization assay or in a microarray. Oligonucleotides may be naturalor synthetic, e.g., DNA, RNA, modified backbones, etc.

Where a particular nucleotide sequence is said to have a specificpercent identity to a reference nucleotide sequence, the percentidentity is relative to the reference nucleotide sequence. For example,a nucleotide sequence that is 50%, 75%, 85%, 90%, 95% or 99% identicalto a reference nucleotide sequence that is 100 bases long can have 50,75, 85, 90, 95 or 99 bases that are completely identical to a 50, 75,85, 90, 95 or 99 nucleotide sequence of the reference nucleotidesequence. The nucleotide sequence can also be a 100 base long nucleotidesequence that is 50%, 75%, 85%, 90%, 95% or 99% identical to thereference nucleotide sequence over its entire length. Of course, thereare other nucleotide sequences that will also meet the same criteria.

A nucleic acid sequence that is “substantially identical” to a VKORnucleotide sequence is at least 80%, 85% 90%, 95% or 99% identical tothe nucleotide sequence of SEQ ID NO:8 or 9. For purposes of comparisonof nucleic acids, the length of the reference nucleic acid sequence willgenerally be at least 40 nucleotides, e.g., at least 60 nucleotides ormore nucleotides. Sequence identity can be measured using sequenceanalysis software (e.g., Sequence Analysis Software Package of theGenetics Computer Group, University of Wisconsin Biotechnology Center,1710 University Avenue, Madison, Wis. 53705).

As is known in the art, a number of different programs can be used toidentify whether a nucleic acid or amino acid has sequence identity orsimilarity to a known sequence. Sequence identity or similarity may bedetermined using standard techniques known in the art, including, butnot limited to, the local sequence identity algorithm of Smith &Waterman, Adv. Appl. Math. 2, 482 (1981), by the sequence identityalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48,443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Natl.Acad. Sci. USA 85,2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Drive, Madison,Wis.), the Best Fit sequence program described by Devereux et al., Nucl.Acid Res. 12, 387-395 (1984), preferably using the default settings, orby inspection.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, J. Mol. Evol.35, 351-360 (1987); the method is similar to that described by Higgins &Sharp, CABIOS 5, 151-153 (1989).

Another example of a useful algorithm is the BLAST algorithm, describedin Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin etal., Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993). A particularlyuseful BLAST program is the WU-BLAST-2 program that was obtained fromAltschul et al., Methods in Enzymology, 266, 460-480 (1996). WU-BLAST-2uses several search parameters, which are preferably set to the defaultvalues. The parameters are dynamic values and are established by theprogram itself depending upon the composition of the particular sequenceand composition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity. An additional useful algorithm is gapped BLAST asreported by Altschul et al Nucleic Acids Res. 25, 3389-3402.

The CLUSTAL program can also be used to determine sequence similarity.This algorithm is described by Higgins et al. (1988) Gene 73:237;Higgins et al. (1989) CABIOS 5:151-153; Corpet et a. (1988) NucleicAcids Res. 16: 10881-90; Huang et al. (1992) CABIOS 8: 155-65; andPearson et al. (1994) Meth. Mol. Biol. 24: 307-331.

In addition, for sequences that contain either more or fewer nucleotidesthan the nucleic acids disclosed herein, it is understood that in oneembodiment, the percentage of sequence identity will be determined basedon the number of identical nucleotides in relation to the total numberof nucleotide bases. Thus, for example, sequence identity of sequencesshorter than a sequence specifically disclosed herein will be determinedusing the number of nucleotide bases in the shorter sequence, in oneembodiment. In percent identity calculations, relative weight is notassigned to various manifestations of sequence variation, such as,insertions, deletions, substitutions, etc.

The VKOR polypeptides of the invention include, but are not limited to,recombinant polypeptides, synthetic peptides and natural polypeptides.The invention also encompasses nucleic acid sequences that encode formsof VKOR polypeptides in which naturally occurring amino acid sequencesare altered or deleted. Preferred nucleic acids encode polypeptides thatare soluble under normal physiological conditions. Also within theinvention are nucleic acids encoding fusion proteins in which all or aportion of VKOR is fused to an unrelated polypeptide (e.g., a markerpolypeptide or a fusion partner) to create a fusion protein. Forexample, the polypeptide can be fused to a hexa-histidine tag tofacilitate purification of bacterially expressed polypeptides, or to ahemagglutinin tag to facilitate purification of polypeptides expressedin eukaryotic cells, or to an HPC4 tag to facilitate purification ofpolypeptides by affinity chromatography or immunoprecipitation. Theinvention also includes isolated polypeptides (and the nucleic acidsthat encode these polypeptides) that include a first portion and asecond portion; the first portion includes, e.g., all or a portion of aVKOR polypeptide, and the second portion includes, e.g., a detectablemarker.

The fusion partner can be, for example, a polypeptide that facilitatessecretion, e.g., a secretory sequence. Such a fused polypeptide istypically referred to as a preprotein. The secretory sequence can becleaved by the cell to form the mature protein. Also within theinvention are nucleic acids that encode VKOR fused to a polypeptidesequence to produce an inactive preprotein. Preproteins can be convertedinto the active form of the protein by removal of the inactivatingsequence.

The invention also includes nucleic acids that hybridize, e.g., understringent hybridization conditions (as defined herein) to all or aportion of the nucleotide sequence of SEQ ID NOS: 1-6, 8 or 9 or theircomplements. In particular embodiments, the hybridizing portion of thehybridizing nucleic acid is typically at least 15 (e.g., 20, 30, or 50)nucleotides in length. The hybridizing portion of the hybridizingnucleic acid is at least 80%, e.g., at least 95%, at least 98% or 100%,identical to the sequence of a portion or all of a nucleic acid encodinga VKOR polypeptide. Hybridizing nucleic acids of the type describedherein can be used, for example, as a cloning probe, a primer (e.g., aPCR primer), or a diagnostic probe. Also included within the inventionare small inhibitory RNAs (siRNAs) and/or antisense RNAs that inhibitthe function of VKOR, as determined, for example, in an activity assay,as described herein and as is known in the art.

In another embodiment, the invention features cells, e.g., transformedcells, that contain a nucleic acid of this invention. A “transformedcell” is a cell into which (or into an ancestor of which) has beenintroduced, by means of recombinant nucleic acid techniques, a nucleicacid encoding all or a part of a VKOR polypeptide, and/or an antisensenucleic acid or siRNA. Both prokaryotic and eukaryotic cells areincluded, e.g., bacteria, yeast, insect, mouse, rat, human, plant andthe like.

The invention also features nucleic acid constructs (e.g., vectors andplasmids) that include a nucleic acid of the invention that is operablylinked to a transcription and/or translation control elements to enableexpression, e.g., expression vectors. By “operably linked” is meant thata selected nucleic acid, e.g., a DNA molecule encoding a VKORpolypeptide, is positioned adjacent to one or more regulatory elements,e.g., a promoter, which directs transcription and/or translation of thesequence such that the regulatory elements can control transcriptionand/or translation of the selected nucleic acid.

The present invention further provides fragments or oligonucleotides ofthe nucleic acids of this invention, which can be used as primers orprobes. Thus, in some embodiments, a fragment or oligonucleotide of thisinvention is a nucleotide sequence that is at least 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,1000, 1500, 2000, 2500 or 3000 contiguous nucleotides of the nucleotidesequence set forth in SEQ ID NO:8 or SEQ ID NO:9. Examples ofoligonucleotides of this invention are provided in the Sequence Listingincluded herewith. Such fragments or oligonucleotides can be detectablylabeled or modified, for example, to include and/or incorporate arestriction enzyme cleavage site when employed as a primer in anamplification (e.g., PCR) assay.

The invention also features purified or isolated VKOR polypeptides, suchas, for example, a polypeptide comprising, consisting essentially ofand/or consisting of the amino acid sequence of SEQ ID NO:10 or abiologically active fragment or peptide thereof. Such fragments orpeptides are typically at least about ten amino acids of the amino acidsequence of SEQ ID NO:10 (e.g., 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 75, 85, 95, 100, 125, or 150 amino acids of the amino acid sequenceof SEQ ID NO:10) and can be peptides or fragment of contiguous aminoacids of the amino acid sequence of the VKOR protein (e.g., as setforthin SEQ ID NO:10). The biological activity of a fragment or peptide ofthis invention can be determined according to the methods providedherein and as are known in the art for identifying VKOR activity. Thefragments and peptides of the VKOR protein of this invention can also beactive as antigens for the production of antibodies. The identificationof epitopes on a fragment or peptide of this invention is carried out bywell known protocols and would be within the ordinary skill of one inthe art.

As used herein, both “protein” and “polypeptide” mean any chain of aminoacids, regardless of length or post-translational modification (e.g.,glycosylation, phosphorylation or N-myristylation). Thus, the term “VKORpolypeptide” includes full-length, naturally occurring VKOR proteins,respectively, as well as recombinantly or synthetically producedpolypeptides that correspond to a full-length, naturally occurring VKORprotein, or to a portion of a naturally occurring or synthetic VKORpolypeptide.

A “purified” or “isolated” compound or polypeptide is a composition thatis at least 60% by weight the compound of interest, e.g., a VKORpolypeptide or antibody that is separated or substantially free from atleast some of the other components of the naturally occurring organismor virus, for example, the cell or viral structural components or otherpolypeptides or nucleic acids commonly found associated with thepolypeptide. As used herein, the “isolated” polypeptide is at leastabout 25%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or morepure (w/w). Preferably the preparation is at least 75% (e.g., at least90% or 99%) by weight the compound of interest. Purity can be measuredby any appropriate standard method, e.g., column chromatography,polyacrylamide gel electrophoresis, or HPLC analysis.

Preferred VKOR polypeptides include a sequence substantially identicalto all or a portion of a naturally occurring VKOR polypeptide.Polypeptides “substantially identical” to the VKOR polypeptide sequencesdescribed herein have an amino acid sequence that is at least 80% or 85%(e.g., 90%, 95% or 99%) identical to the amino acid sequence of the VKORpolypeptides of SEQ ID NO: 10. For purposes of comparison, the length ofthe reference VKOR polypeptide sequence will generally be at least 16amino acids, e.g., at least 20, 25, 30, 35, 40, 45, 50, 75, or 100 aminoacids.

In the case of polypeptide sequences that are less than 100% identicalto a reference sequence, the non-identical positions are preferably, butnot necessarily, conservative substitutions for the reference sequence.Conservative substitutions typically include, but are not limited to,substitutions within the following groups: glycine and alanine; valine,isoleucine, and leucine; aspartic acid and glutamic acid; asparagine andglutamine; serine and threonine; lysine and arginine; and phenylalanineand tyrosine.

Where a particular polypeptide is said to have a specific percentidentity to a reference polypeptide of a defined length, the percentidentity is relative to the reference polypeptide. Thus, for example, apolypeptide that is 50%, 75%, 85%, 90%, 95% or 99% identical to areference polypeptide that is 100 amino acids long can be a 50, 75, 85,90, 95 or 99 amino acid polypeptide that is completely identical to a50, 75, 85, 90, 95 or 99 amino acid long portion of the referencepolypeptide. It can also be a 100 amino acid long polypeptide that is50%, 75%, 85%, 90%, 95% or 99% identical to the reference polypeptideover its entire length. Of course, other polypeptides also will meet thesame criteria.

The invention also features purified or isolated antibodies thatspecifically bind to a VKOR polypeptide of this invention or to afragment thereof. By “specifically binds” is meant that an antibodyrecognizes and binds a particular antigen, e.g., a VKOR polypeptide, oran epitope on a fragment or peptide of a VKOR polypeptide, but does notsubstantially recognize and bind other molecules in a sample. In oneembodiment the antibody is a monoclonal antibody and in otherembodiments, the antibody is a polyclonal antibody. The production ofboth monoclonal and polyclonal antibodies, including chimericantibodies, humanized antibodies, single chain antibodies, bi-specificantibodies, antibody fragments, etc., is well known in the art.

In another aspect, the invention features a method for detecting a VKORpolypeptide in a sample. This method comprises contacting the samplewith an antibody that specifically binds a VKOR polypeptide or afragment thereof under conditions that allow the formation of a complexbetween an antibody and VKOR; and detecting the formation of a complex,if any, as detection of a VKOR polypeptide or fragment thereof in thesample. Such immunoassays are well known in the art and includeimmunoprecipitation assays, immunoblotting assays, immunolabelingassays, ELISA, etc.

The present invention further provides a method of detecting a nucleicacid encoding a VKOR polypeptide in a sample, comprising contacting thesample with a nucleic acid of this invention that encodes VKOR or afragment thereof, or a complement of a nucleic acid that encodes VKOR ora fragment thereof, under conditions whereby a hybridization complex canform, and detecting formation of a hybridization complex, therebydetecting a nucleic acid encoding a VKOR polypeptide in a sample. Suchhybridization assays are well known in the art and include probedetection assays and nucleic acid amplification assays.

Also encompassed by the invention is a method of obtaining a generelated to (i.e., a functional homologue of) the VKOR gene. Such amethod entails obtaining or producing a detectably-labeled probecomprising an isolated nucleic acid which encodes all or a portion ofVKOR, or a homolog thereof; screening a nucleic acid fragment librarywith the labeled probe under conditions that allow hybridization of theprobe to nucleic acid fragments in the library, thereby forming nucleicacid duplexes; isolating labeled duplexes, if any; and preparing afull-length gene sequence from the nucleic acid fragments in any labeledduplex to obtain a gene related to the VKOR gene.

A further aspect of the present invention is a method of making avitamin K dependent protein which comprises culturing a host cell thatexpresses a nucleic acid encoding a vitamin K dependent protein in thepresence of vitamin K and produces a vitamin K dependent protein, andthen harvesting the vitamin K dependent protein from the culture, thehost cell containing and expressing a heterologous nucleic acid encodingvitamin K dependent carboxylase, and the host cell further containingand expressing a heterologous nucleic acid encoding vitamin K epoxidereductase (VKOR) and producing VKOR as described herein. The expressionof the VKOR-encoding nucleic acid and the production of the VKOR causesthe cell to produce greater levels of the vitamin K dependent proteinthan would be produced in the absence of the VKOR.

Thus, in some embodiments, the present invention also provides a methodof producing a vitamin K dependent protein, comprising:

a) introducing into a cell a nucleic acid that encodes a vitamin Kdependent protein under conditions whereby the nucleic acid is expressedand the vitamin K dependent protein is produced in the presence ofvitamin K, wherein the cell comprises a heterologous nucleic acidencoding vitamin K dependent carboxylase and further comprises aheterologous nucleic acid encoding vitamin K epoxide reductase; and

b) optionally collecting the vitamin K dependent protein from the cell.The vitamin K dependent protein that can be produced can be any vitaminK dependent protein now known or later identified as such, including butnot limited to Factor VII, Factor IX, Factor X, Protein C, Protein S andprothrombin, in any combination. Any host cell that can be transformedwith the nucleic acids described can be used as described herein,although in some embodiments non-human or even non-mammalian host cellscan be used. Nucleic acids encoding vitamin K dependent carboxylase andnucleic acids encoding vitamin K dependent proteins as described hereinare well known in the art and their introduction into cells forexpression would be carried out according to routine protocols.

Certain embodiments of this invention are based on the inventors'discovery that a subject's therapeutic dose of warfarin foranticoagulation therapy can be correlated with the presence of one ormore single nucleotide polymorphisms in the VKOR gene of the subject.Thus, the present invention also provides a method of identifying ahuman subject having increased or decreased sensitivity to warfarin,comprising detecting in the subject the presence of a single nucleotidepolymorphism (SNP) in the VKOR gene, wherein the single nucleotidepolymorphism is correlated with increased or decreased sensitivity towarfarin, thereby identifying the subject as having increased ordecreased sensitivity to warfarin.

An example of a SNP correlated with an increased sensitivity to warfarinis a G→C alteration at nucleotide 2581 (SEQ ID NO:12) (in intron 2 ofthe VKOR gene; GenBank accession no. refSNP ID: rs8050894, incorporatedby reference herein) of the nucleotide sequence of SEQ ID NO:11, whichis a reference sequence encompassing the genomic sequence of SEQ ID NO:8and approximately 1000 nucleotides preceding and following thissequence. This sequence can be located as having the genome position“human chromosome 16p11.2” or in the physical map in the NCBI databaseas human chromosome 16: 31009700-31013800.

Examples of SNPs correlated with a decreased sensitivity to warfarin area T→C alteration at nucleotide 3294 (SEQ ID NO:13) (in intron 2 of theVKOR gene; GenBank accession no. refSNP ID: rs2359612, incorporated byreference herein) of the nucleotide sequence of SEQ ID NO:11 and a G→Aalteration at nucleotide 4769 (SEQ ID NO:14) (in the 3′ UTR of the VKORgene; GenBank accession no. refSNP ID: rs7294, incorporated by referenceherein) of the nucleotide sequence of SEQ ID NO:11.

As used herein, a subject having an “increased sensitivity to warfarin”is a subject for whom a suitable therapeutic or maintenance dose ofwarfarin is lower than the therapeutic or maintenance dose of warfarinthat would suitable for a normal subject, i.e., a subject who did notcarry a SNP in the VKOR gene that imparts a phenotype of increasedsensitivity to warfarin. Conversely, as used herein, a subject having a“decreased sensitivity to warfarin” is a subject for whom a suitabletherapeutic or maintenance dose of warfarin is higher than thetherapeutic or maintenance dose of warfarin that would suitable for anormal subject, i.e., a subject who did not carry a SNP in the VKOR genethat imparts a phenotype of decreased sensitivity to warfarin. Anexample of a typical therapeutic dose of warfarin for a normal subjectis 35 mg per week, although this amount can vary (e.g., a dose range of3.5 to 420 mg per week is described in Aithal et al. (1999) Lancet353:717-719). A typical therapeutic dose of warfarin can be determinedfor a given study group according to the methods described herein, whichcan be used to identify subjects with therapeutic warfarin doses aboveor below this dose, thereby identifying subjects having decreased orincreased sensitivity to warfarin.

Further provided herein is a method of identifying a human subjecthaving increased or decreased sensitivity to warfarin, comprising: a)correlating the presence of a single nucleotide polymorphism in the VKORgene with increased or decreased sensitivity to warfarin; and b)detecting the single nucleotide polymorphism of step (a) in the subject,thereby identifying a subject having increased or decreased sensitivityto warfarin.

In addition, the present invention provides a method of identifying asingle nucleotide polymorphism in the VKOR gene correlated withincreased or decreased sensitivity to warfarin, comprising: a)identifying a subject having increased or decreased sensitivity towarfarin; b) detecting in the subject the presence of a singlenucleotide polymorphism in the VKOR gene; and c) correlating thepresence of the single nucleotide polymorphism of step (b) with theincreased or decreased sensitivity to warfarin in the subject, therebyidentifying a single nucleotide polymorphism in the VKOR gene correlatedwith increased or decreased sensitivity to warfarin.

Also provided herein is a method of correlating a single nucleotidepolymorphism in the VKOR gene of a subject with increased or decreasedsensitivity to warfarin, comprising: a) identifying a subject havingincreased or decreased sensitivity to warfarin; b) determining thenucleotide sequence of the VKOR gene of the subject of (a); c) comparingthe nucleotide sequence of step (b) with the wild type nucleotidesequence of the VKOR gene; d) detecting a single nucleotide polymorphismin the nucleotide sequence of (b); and e) correlating the singlenucleotide polymorphism of (d) with increased or decreased sensitivityto warfarin in the subject of (a).

A subject is identified as having an increased or decreased sensitivityto warfarin by establishing a therapeutic or maintenance dose ofwarfarin for anticoagulation therapy according to well known protocolsand comparing the therapeutic or maintenance dose for that subject withthe therapeutic or maintenance dose of warfarin for anticoagulationtherapy of a population of normal subjects (e.g., subjects lacking anySNPs in the VKOR gene correlated with increased or decreased sensitivityto warfarin) from which an average or mean therapeutic or maintenancedose of warfarin is calculated. A subject having a therapeutic ormaintenance dose of warfarin that is below the average therapeutic ormaintenance dose of warfarin (e.g., the dose of warfarin that istherapeutic or provides a maintenance level for a subject that has awild type VKOR gene, i.e., lacking any single nucleotide polymorphismsassociated with warfarin sensitivity) is a subject identified as havingan increased sensitivity to warfarin. A subject having a therapeutic ormaintenance dose of warfarin that is above the average therapeutic ormaintenance of warfarin is a subject identified as having a decreasedsensitivity to warfarin. An average therapeutic or maintenance dose ofwarfarin for a subject with a wild type VKOR gene would be readilydetermined by one skilled in the art.

The nucleotide sequence of the VKOR gene of a subject is determinedaccording to methods standard in the art, and as described in theExamples provided herein. For example, genomic DNA is extracted fromcells of a subject and the VKOR gene is located and sequenced accordingto known protocols. Single nucleotide polymorphisms in the VKOR gene areidentified by a comparison of a subject's sequence with the wild typesequence as known in the art (e.g., the reference sequence as shownherein as SEQ ID NO:11).

A SNP in the VKOR gene is correlated with an increased or decreasedsensitivity to warfarin by identifying the presence of a SNP or multipleSNPs in the VKOR gene of a subject also identified as having increasedor decreased sensitivity to warfarin, i.e., having a maintenance ortherapeutic dose of warfarin that is above or below the average dose andperforming a statistical analysis of the association of the SNP or SNPswith the increased or decreased sensitivity to warfarin, according towell known methods of statistical analysis. An analysis that identifiesa statistical association (e.g., a significant association) between theSNP(s) (genotype) and increased or decreased warfarin sensitivity(phenotype) establishes a correlation between the presence of the SNP(s)in a subject and an increased or decreased sensitivity to warfarin inthat subject.

It is contemplated that a combination of factors, including the presenceof one or more SNPs in the VKOR gene of a subject, can be correlatedwith an increased or decreased sensitivity to warfarin in that subject.Such factors can include, but are not limited to cytochrome p450 2C9polymorphisms, race, age, gender, smoking history and hepatic disease.

Thus, in a further embodiment, the present invention provides a methodof identifying a human subject having increased or decreased sensitivityto warfarin, comprising identifying in the subject the presence of acombination of factors correlated with an increased or decreasedsensitivity to warfarin selected from the group consisting of one ormore single nucleotide polymorphisms of the VKOR gene, one or morecytochrome p450 2C9 polymorphisms, race, age, gender, smoking history,hepatic disease and any combination of two or more of these factors,wherein the combination of factors is correlated with increased ordecreased sensitivity to warfarin, thereby identifying the subjecthaving increased or decreased sensitivity to warfarin.

Further provided herein is a method of identifying a human subjecthaving increased or decreased sensitivity to warfarin, comprising: a)correlating the presence of a combination of factors with an increasedor decreased sensitivity to warfarin, wherein the factors are selectedfrom the group consisting of one or more single nucleotide polymorphismsof the VKOR gene, one or more cytochrome p450 2C9 polymorphisms, race,age, gender, smoking history, hepatic disease and any combination of twoor more of these factors; and b) detecting the combination of factors ofstep (a) in the subject, thereby identifying a subject having increasedor decreased sensitivity to warfarin.

In addition, the present invention provides a method of identifying acombination of factors correlated with an increased or decreasedsensitivity to warfarin, wherein the factors are selected from the groupconsisting of one or more single nucleotide polymorphisms of the VKORgene, one or more cytochrome p450 2C9 polymorphisms, race, age, gender,smoking history, hepatic disease and any combination of two or more ofthese factors, comprising: a) identifying a subject having increased ordecreased sensitivity to warfarin; b) detecting in the subject thepresence of a combination of the factors; and c) correlating thepresence of the combination of factors of step (b) with the increased ordecreased sensitivity to warfarin in the subject, thereby identifying acombination of factors correlated with increased or decreasedsensitivity to warfarin.

Also provided herein is a method of correlating a combination offactors, wherein the factors are selected from the group consisting ofone or more single nucleotide polymorphisms of the VKOR gene, one ormore cytochrome p450 2C9 polymorphisms, race, age, gender, smokinghistory, hepatic disease and any combination of two or more of thesefactors, with increased or decreased sensitivity to warfarin,comprising: a) identifying a subject having increased or decreasedsensitivity to warfarin; b) identifying the presence of a combination ofthe factors in the subject; and c) correlating the combination of thefactors of (b) with increased or decreased sensitivity to warfarin inthe subject of (a).

A combination of factors as described herein is correlated with anincreased or decreased sensitivity to warfarin by identifying thepresence of the combination of factors in a subject also identified ashaving increased or decreased sensitivity to warfarin and performing astatistical analysis of the association of the combination of factorswith the increased or decreased sensitivity to warfarin, according towell known methods of statistical analysis. An analysis that identifiesa statistical association (e.g., a significant association) between thecombination of factors and the warfarin sensitivity phenotype (increasedor decreased) establishes a correlation between the presence of thecombination of factors in a subject and an increased or decreasedsensitivity to warfarin in that subject.

Further provided herein are nucleic acids encoding VKOR and comprisingone or more SNPs as described herein. Thus, the present inventionfurther provides nucleic acids comprising, consisting essentially ofand/or consisting of the nucleotide sequence as set forth in SEQ IDNOs:12, 13, 14, 15 and 16. The nucleic acids can be present in a vectorand the vector can be present in a cell. Further included are proteinsencoded by a nucleic acid comprising a nucleotide sequence as set forthin SEQ ID NOs:12, 13, 14,15 and 16, as well as antibodies thatspecifically bind a protein encoded by a nucleic acid comprising anucleotide sequence as set forth in SEQ ID NOs:12, 13, 14, 15 and 16.The present invention is more particularly described in the followingexamples that are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

EXAMPLES Example I Correlation Between SNPS in VKOR Gene and Increasedor Decreased Sensitivity to Warfarin

The most prevalent isoform of the VKOR gene is about 4 kb long, hasthree exons and encodes an enzyme of 163 amino acids with a mass of 18.4kDa. In the present study, three mutations vk2581 (G>C), vk3294(T>C) andvk4769(G>A), identified as SNPs (heterozygosity ratios of 46.9%, 46.8%and 46.3%, respectively) were examined for a correlation between theirpresence in a subject and the maintenance dose of warfarin required toachieve a therapeutically effective response.

1. Selection of Subjects

Subjects were obtained from the UNC Coagulation Clinic in the AmbulatoryCare Center. Informed consent was obtained by a trained geneticcounselor. Subjects not fluent in English were excluded because of thelack of translators and the requirement for consent. To qualify for thestudy, subjects had warfarin for at least six months, were older than 18and were followed by the UNC Coagulation clinic at the Ambulatory CareClinic.

2. Extraction of Genomic DNA from Whole Blood

Genomic DNAs were extracted from the whole blood of subjects usingQIAamp DNA Blood Mini Kit (QIAGEN cat#51104). The DNA concentration wasadjusted to 10 ng/μL.

3. Sequencing of the Genomic DNA Samples

Approximately 10 ng of DNA was used for polymerase chain reaction (PCR)assays. The primers used to amplify the VKOR gene were: Exon 1-5′CCAATCGCCGAGTCAGAGG (SEQ ID NO:29) and Exon 1-3′ CCCAGTCCCCAGCACTGTCT(SEQ ID NO:30) for the 5′-UTR and Exon 1 region; Exon 2-5′AGGGGAGGATAGGGTCAGTG (SEQ ID NO:31) and Exon 2-3′ CCTGTTAGTTACCTCCCCACA(SEQ ID NO:32) for the Exon 2 region; and Exon 3-5′ ATACGTGCGTAAGCCACCAC(SEQ ID NO:33) and Exon 3-3′ ACCCAGATATGCCCCCTTAG (SEQ ID NO:34) for theExon3 and 3′-UTR region. Automated high throughput capillaryelectrophoresis DNA sequencing was used for detecting SNPs in the VKORgene.

4. Detection of Known SNPs using Real-time PCR

The assay reagents for SNP genotyping were from the Assay-by-Design™service (Applied Biosystems, cat#4332072). The primers and probes (FAM™and VIC™ dye-labeled) were designed using Primer Express software andwere synthesized in an Applied Biosystems synthesizer. The primer pairsfor each SNP are located at the upstream/downstream position of the SNPsite and can generate less than 100 bp length of a DNA fragment in thePCR reaction. The FAM™ and VIC™ dye-labeled probes were designed tocover the SNP sites with a length of 15-16 nt. The primer and probesequences for each VKOR SNP are shown in Table 2.

The 2× TaqMan™ Universal PCR Master Mix, No AmpErase UNG (AppliedBiosystems, cat#4324018) was used in the PCR reactions. Forty cycles ofreal-time PCR were performed in an Opticon II (MJ Research) machine.There was a 10 minute 95° C. preheat followed by 92° C. for 15 sec, 60°C. for 1 min. and then a plate reading. The results were read accordingto the signal value of FAM and VIC dye.

5. Statistical Analysis

The difference of average dose between different genotypes was comparedby analysis of variance (ANOVA) using SAS version 8.0 (SAS, Inc., Cary,N.C.). A two-sided p value less than 0.05 was considered significant.Examination of the distribution and residuals for the average dose oftreatment among the SNP groups indicated that a log transformation wasnecessary to satisfy the assumption of homogeneity of variance.

6. Correlation of SNPs with Warfarin Dosage

By direct genomic DNA sequencing and SNP real-time PCR detection, fiveSNPs were identified in the VKOR gene: one in the 5′-UTR, two in intron11, one in the coding region and one in the 3′-UTR (Table 1).

Among these SNPs, the vk563 and vk4501 SNPs allele were carried by onlyone of the 58 subjects of the study (a triple heterozygous, alsocarrying the 3′-UTR SNP allele), while the other SNPs were identified in17-25 heterozygous patients.

Each marker was first analyzed independently. FIG. 1A shows that theaverage warfarin dose for patients with the vk2581 wild type allele was50.19±3.20 mg per week (n=26), while those heterozygous and homozygousfor this polymorphism were 35.19±3.73 (n=17) and 31.14±6.2 mg per week(n=15), respectively. FIG. 1B shows that the average warfarin dose forpatients with the wild-type vk3294 allele was 25.29±3.05 mg per week(n=11), while patients bearing the heterozygous and homozygous alleleswere 41.68±4.92 (n=25) and 47.73±2.75 mg per week (n=22), respectively.FIG. 1C shows the average warfarin dose for patients with vk4769 SNPwild type was 35.35±4.01 mg per week (n=27), while patients with theheterozygous and homozygous alleles required 44.48±4.80 (n=19) and47.56±3.86 mg per week (n=12), respectively. It was also observed thatP450 2C9 *3 has a significant effect on warfarin dose (FIG. 1D), aspreviously reported (Joffe et al. (2004) “Warfarin dosing and cytochromeP450 2C9 polymorphisms” Thromb Haemost 91:1123-1128). The averagewarfarin dose for patients with P450 2C9 *1 (wild type) was 43.82±2.75mg per week (n=50), while patients heterozygous for this allele required22.4±4.34 mg per week (n=8).

7. Statistical Analysis

The association of the Log_(e) (warfarin average dosage)(LnDose) withthe SNPs in the VKOR gene was examined by analysis of variance (ANOVA).SAS was used first to do a repeated procedure in which a series offactors (race, gender, smoking history, hepatic diseases, SNPs atcytochrome P450 2Y9 gene, etc.) were examined to identify factors,excluding VKOR SNPs, which might affect dosage. P450 2C9 *3 wassignificantly associated with the average dose of warfarin; thus, it wasincluded as a covariant for further analysis. The analysis indicatedthat the three VKOR SNPs were still significantly associated with weeklywarfarin dose (vk2581, P<0.0001; vk3294, P<0.0001; and vk4769,P=0.0044), when the covariance is included.

To specifically test if the three SNPs of VKOR were independentlyassociated with warfarin dosage, the analysis was repeated in which twoSNPs in the VKOR gene were included as covariates for the other SNP. Thethree VKOR SNPs are located within 2 kb distance of one another and areexpected to be closely linked. It was clear from inspection that, atleast for Caucasians, one haplotype (where A=vk2581 guanine and a=vk2581cytosine; B=vk3294 thymine and b=vk3924 cytosine; C=vk4769 guanine andc=vk4769 adenine) was Mbbcc and another aaBBCC. The distribution ofindividual SNPs in patients was found to be significantly correlatedwith the others (R=0.63-0.87, p<0.001). Indeed, subjects with thehaplotype AAbbcc (n=7) required a significantly higher dosage ofwarfarin (warfarin dosage=48.98±3.93) compared to those patients withhaplotype aaBBCC (25.29±3.05; p<0.001).

Example 2 siRNA Design and Synthesis

siRNAs were selected using an advanced version of a rational designalgorithm (Reynolds et al. (2004) “Rational siRNA design for RNAinterference” Nature Biotechnology 22:326-330). For each of the 13genes, four siRNAs duplexes with the highest scores were selected and aBLAST search was conducted using the Human EST database. To minimize thepotential for off-target silencing effects, only those sequence targetswith more than three mismatches against un-related sequences wereselected (Jackson et al. (2003) “Expression profiling reveals off-targetgene regulation by RNAi” Nat Biotechnol 21:635-7). All duplexes weresynthesized in Dharmacon (Lafayette, Colo.) as 21-mers with UU overhangsusing a modified method of 2′-ACE chemistry (Scaringe (2000) “Advanced5′-silyl-2′-orthoester approach to RNA oligonucleotide synthesis”Methods Enzymol 317:3-18) and the AS strand was chemicallyphosphorylated to ensure maximum activity (Martinez et al. (2002)“Single-stranded antisense siRNAs guide target RNA cleavage in RNAi”Cell 110:563-74).

Example 3 siRNA Transfection

Transfection was essentially as previously described (Harborth et al.(2001) “Identification of essential genes in cultured mammalian cellsusing small interfering RNAs” J Cell Sci 114:4557-65) with minormodifications.

Example 4 VKOR Activity Assay

siRNA transfected A549 cells were trypsinized and washed twice with coldPBS. 1.5×10⁷ cells were taken for each VKOR assay. 200 μL buffer D (250mM Na₂HPO₄—NaH₂PO₄, 500 mM KCl, 20% glycerol and 0.75% CHAPS, pH 7.4)was added to the cell pellet, followed by sonication of the cell lysate.For assays of solubilized microsomes, microsomes were prepared from2×10⁹ cells as described (Lin et al. (2002) “The putative vitaminK-dependent gamma-glutamyl carboxylase internal propeptide appears to bethe propeptide binding site” J Biol Chem 277:28584-91); 10 to 50 μL ofsolubilized microsomes were used for each assay. Vitamin K epoxide wasadded to the concentration indicated in the figure legends and DTT wasadded to 4 mM to initiate the reaction. The reaction mixture wasincubated in yellow light at 30° C. for 30 minutes and stopped by adding500 μL 0.05 M AgNO₃: isopropanol (5:9). 500 μL hexane was added and themixture was vortexed vigorously for 1 minute to extract the vitamin Kand KO. After 5 minutes centrifugation, the upper organic layer wastransferred to a 5-mL brown vial and dried with N₂. 150 μL HPLC buffer,acetonitrile:isopropanol:water (100:7:2), was added to dissolve thevitamin K and KO and the sample was analyzed by HPLC on an A C-18 column(Vydac, cat#218TP54).

Example 5 RT-qPCR (Reverse Transcriptase Quantitative PCR)

1×10⁶ cells were washed with PBS twice and total RNA was isolated withTrizol reagent according to the manufacturer's protocol (Invitrogen). 1μg of RNA was digested by RQ1 DNasel (Promega) and heat-inactivated.First strand cDNA was made with M-MLV reverse transcriptase(Invitrogen). cDNAs were mixed with DyNAmo SYBR Green qPCR pre-mix(Finnzymes) and real-time PCR was performed with an Opticon II PCRthermal cycler (MJ Research). The following primers were used:

13124769-5′ (F): (TCCAACAGCATATTCGGTTGC, SEQ ID NO: 1); 13124769-3 (R)′:(TTCTTGGACCTTCCGGAAACT, SEQ ID NO: 2); GAPDH-F: (GAAGGTGAAGGTCGGAGTC,SEQ ID NO: 3); GAPDH-R: (GAAGATGGTGATGGGATTTC, SEQ ID NO: 4);Lamin-RT-F: (CTAGGTGAGGCCAAGAAGCAA, SEQ ID NO: 5) and Lamin-RT-R:(CTGTTCCTCTCAGCAGACTGC, SEQ ID NO: 6).

Example 6 Over-expression of VKOR in Sf9 Insect Cell Line

The cDNA for the mGC11276 coding region was cloned into pVL1392(Pharmingen), with the HPC4 tag (EDQVDPRLIDGK, SEQ ID NO: 7) at itsamino terminus and expressed in Sf9 cells as described (Li et al. (2000)“Identification of a Drosophila vitamin K-dependent gamma-glutamylcarboxylase” J Biol Chem 275:18291-6).

Example 7 Gene Selection

The search for the VKOR gene was focused on human chromosome sixteenbetween markers D16S3131 and D16S419. This region corresponds tochromosome 16 at 50 cM-65 cM on the genetic map and 2646.3 Mb on thephysical map. 190 predicted coding sequences in this region wereanalyzed by a BLASTX search of the NCBI non-redundant protein database.Those human genes and orthologs from related species with known functionwere eliminated. Because VKOR appears to be a transmembrane protein(Carlisle & Suttie (1980) “Vitamin K dependent carboxylase: subcellularlocation of the carboxylase and enzymes involved in vitamin K metabolismin rat liver” Biochemistry 19:1161-7), the remaining genes weretranslated according to the cDNA sequences in the NCBI database andanalyzed with the programs TMHMM and TMAP (Biology WorkBench, San DiegoSupercomputer System) to predict those with transmembrane domains.Thirteen genes predicted to code for integral membrane proteins werechosen for further analysis.

Example 8 Cell Line Screening for VKOR Activity

The strategy was to identify a cell line expressing relatively highamounts of VKOR activity and use siRNA to systematically knock down allthirteen candidate genes. siRNA, double stranded RNA of 21-23nucleotides, has been shown to cause specific RNA degradation in cellculture (Hara et al. (2002) “Raptor, a binding partner of target ofrapamycin (TOR), mediates TOR action” Cell 110:177-89; Krichevsky &Kosik (2002) “RNAi functions in cultured mammalian neurons” Proc NatlAcad Sci USA 99:11926-9; Burns et al. (2003) “Silencing of the Novel p53Target Gene Snk/Plk2 Leads to Mitotic Catastrophe in Paclitaxel(Taxol)-Exposed Cells” Mol Cell Biol 23:5556-71). However, applicationof siRNA for large scale screening in mammalian cells has not previouslybeen reported because of the difficulty in identifying a functionaltarget for a specific mammalian cell mRNA (Holen et al. (2003) “Similarbehaviour of single-strand and double-strand siRNAs suggests they actthrough a common RNAi pathway” Nucleic Acids Res 31:2401-7). Thedevelopment of a rational selection algorithm (Reynolds et al.) forsiRNA design increases the probability that a specific siRNA can bedeveloped; furthermore, the probability of success can be increased bypooling four rationally selected siRNAs. Using siRNA to search forpreviously unidentified genes has the advantage that, even if VKORactivity requires the product of more than one gene for activity, thescreen should still be effective because the assay determines the lossof enzymatic activity.

Fifteen cell lines were screened and a human lung carcinoma line, A549,was identified to exhibit sufficient warfarin-sensitive VKOR activityfor facile measurement. A second human colorectal adenocarcinoma cellline, HT29, which expressed very little VKOR activity, was used as areference.

Example 9 siRNA Inhibition of VKOR Activity in A549 Cells

Each of the thirteen pools of siRNA were transfected in triplicate intoA549 cells and assayed for VKOR activity after 72 hours. One siRNA poolspecific for gene gi:13124769 reduced VKOR activity by 64%-70% in eightseparate assays (FIG. 2).

One possible reason that VKOR activity was inhibited to only ˜35% of itsinitial activity after 72 hours is that the half-life of mammalianproteins varies greatly (from minutes to days) (Zhang et al. (1996) “Themajor calpain isozymes are long-lived proteins. Design of an antisensestrategy for calpain depletion in cultured cells” J Biol Chem271:18825-30; Bohley (1996) “Surface hydrophobicity and intracellulardegradation of proteins” Biol Chem 377:425-35; Dice & Goldberg (1975)“Relationship between in vivo degradative rates and isoelectric pointsof proteins” Proc Natl Acad Sci USA 72:3893-7), and mRNA translation isbeing inhibited, not enzyme activity. Therefore, the cells were carriedthrough eleven days and their VKOR activity followed. FIG. 3 shows thatthe level of mRNA for gi:13124769 mRNA decreased rapidly to about 20% ofnormal while VKOR activity decreased continuously during this timeperiod. This reduction in activity is not a general effect of the siRNAor the result of cell death because the level of VKD carboxylaseactivity and lamin A/C mRNA remained constant. Furthermore, the level ofgi:132124769 mRNA is four fold lower in HT-29 cells, which have low VKORactivity, than in A549 cells that exhibit high VKOR activity. These dataindicate that gi:13124769 corresponds to the VKOR gene.

Example 10 Identification of Gene Encoding VKOR

The gene, IMAGE 3455200 (gi:13124769, SEQ ID NO: 8), identified hereinto encode VKOR, maps to human chromosome 16p11.2, mouse chromosome 7F3,and rat chromosome 1:180.8 Mb. There are 338 cDNA clones in the NCBIdatabase representing seven different splicing patterns (NCBI AceViewprogram). These are composed of all or part of two to four exons. Amongthese, the most prevalent isoform, mGC11276, has three exons and isexpressed at high levels in lung and liver cells. This three exontranscript (SEQ ID NO: 9) encodes a predicted protein of 163 amino acidswith a mass of 18.2 kDa (SEQ ID NO: 10). It is a putative N-myristylatedendoplasmic reticulum protein with one to three transmembrane domains,depending upon the program used for prediction. It has seven cysteineresidues, which is consistent with observations that the enzymaticactivity is dependent upon thiol reagents (Thijssen et al. (1994)“Microsomal lipoamide reductase provides vitamin K epoxide reductasewith reducing equivalents” Biochem J 297:277-80). Five of the sevencysteines are conserved among human, mice, rat, zebrafish, Xenopus andAnopheles.

To confirm that the VKOR gene had been identified, the most prevalentform of the enzyme (three exons) was expressed in Spodoptera frugiperda,Sf9 cells. Sf9 cells have no measurable VKOR activity but exhibitwarfarin sensitive activity when transfected with mGC11276 cDNA (FIG.4). VKOR activity is observed from constructs with an epitope tag ateither their amino or carboxyl terminus. This tag should assist in thepurification of VKOR.

VKOR should exhibit warfarin sensitivity, therefore microsomes were madefrom Sf9 cells expressing VKOR and tested for warfarin sensitivity. TheVKOR activity is warfarin-sensitive (FIG. 5).

In summary, the present invention provides the first example of usingsiRNA in mammalian cells to identify an unknown gene. The identity ofthe VKOR gene was confirmed by its expression in insect cells. The VKORgene encodes several isoforms. It will be important to characterize theactivity and expression pattern of each isoform. Millions of peopleworld-wide utilize warfarin to inhibit coagulation; therefore it isimportant to further characterize VKOR as it can lead to more accuratedosing or design of safer, more effective, anti-coagulants.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

All publications, patent applications, patents, patent publications andother references cited herein are incorporated by reference in theirentireties for the teachings relevant to the sentence and/or paragraphin which the reference is presented.

TABLE 1 Five SNPs examined in VKOR gene SNPs position AA changeHeterozygous ratio vk563 5′- N/A  1/58 G > A UTR (SEQ ID NO: 15) vk2581G > C Intron2 N/A 17/58 (SEQ ID NO: 12) vk3294 T > C Intron2 N/A 25/58(SEQ ID NO: 13) vk4501 C > T Exon3 Leu120Leu  1/58 (SEQ ID NO: 16 vk4769G > A 3′- N/A 19/58 (SEQ ID UTR NO: 14

TABLE 2 VIC Probe FAM Probe SNPs Sequence Sequence Forward PrimerReverse Primer vk2581 TCATCACGGAGCGTC TCATCACCGAGCGTCGGTGATCCACACAGCTGACA CCTGTTAGTTACCTCCCCACATC G > C (SEQ ID NO:17) (SEQID NO:18) (SEQ ID NO:19) (SEQ ID NO:20) vk3294 CCAGGACCATGGTGCCCAGGACCGTGGTGC GCTCCAGAGAAGGCATCACT GCCAAGTCTGAACCATGTGTCA T > C (SEQID NO:21) (SEQ ID NO:22) (SEQ ID NO:23) (SEQ ID NO:24) vk4769ATACCCGCACATGAC CATACCCACACATGAC GTCCCTAGAAGGCCCTAGATGTGTGTGGCACATTTGGTCCATT G > A (SEQ ID NO:25) (SEQ ID NO:26) (SEQ ID NO:27)(SEQ ID NO:28)

1. A method of amplifying a segment of a VKOR genomic nucleotidesequence, comprising: a) choosing a first oligonucleotide primer fromthe 3′ end of a nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO:8; b) choosing a second oligonucleotide primer from the 5′end of a nucleic acid molecule comprising the nucleotide sequence of SEQID NO:8; c) adding said first primer and said second primer to a nucleicacid sample; and d) amplifying a segment of the VKOR genomic nucleotidesequence defined by the first primer and the second primer, wherein saidnucleic acid sample is from a subject in need of warfarin therapy. 2.The method of claim 1, wherein the amplified segment of step (d) is lessthan 100 base pairs in length.
 3. The method of claim 1, wherein theamplified segment of step (d) comprises a single nucleotidepolymorphism.
 4. The method of claim 1, wherein the firstoligonucleotide primer is at least 15 nucleotides in length.
 5. Themethod of claim 1, wherein the second oligonucleotide primer is at least15 nucleotides in length.
 6. A method of amplifying a segment of a VKORgenomic nucleotide sequence, comprising: a) choosing a firstoligonucleotide primer from a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:8; b) choosing a second oligonucleotideprimer from a nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO:8 that differs in nucleotide sequence from the firstoligonucleotide primer; c) adding said first primer and said secondprimer to a nucleic acid sample; and d) amplifying a segment of the VKORgenomic nucleotide sequence defined by the first primer and the secondprimer, wherein said nucleic acid sample is from a subject in need ofwarfarin therapy.
 7. The method of claim 6, wherein the amplifiedsegment of step (d) is less than 100 base pairs in length.
 8. The methodof claim 6, wherein the amplified segment of step (d) comprises a singlenucleotide polymorphism.
 9. The method of claim 6, wherein the firstoligonucleotide primer is at least 15 nucleotides in length.
 10. Themethod of claim 6, wherein the second oligonucleotide primer is at least15 nucleotides in length.